Lantibiotics are post-translationally modified peptide antibiotics. Nisin, its best known member, has been used for decades in the food industry without significant development of resistance. Other lantibiotics are under investigation or in clinical trials for the treatment of cystic fibrosis and asthma, the elimination of tooth-decay causing bacteria, and for the treatment of multi-drug resistant bacteria. In this grant application we seek to extend our past success on the use of the biosynthetic machinery to make analogs and to better understand their mechanism of substrate recognition and catalysis. Our goals are: 1. Obtain co-crystal structures of the lantibiotic synthetases with their substrates and carry out binding studies to define their mechanism of substrate recognition and activation. 2. Obtain in depth kinetic information on the dehydration and cyclization steps, determine the processivity of these processes, and elucidate the order of ring formation. 3. Use of the biosynthetic machinery for SAR studies on haloduracin and to develop general tools for protein chemistry. 4. Investigate the biosynthetic pathway for new lantibiotics including duramycin and cinnamycin. PUBLIC HEALTH RELEVANCE Numerous reports of multi-drug resistant bacterial strains have appeared in recent years, with several strains posing the serious threat of becoming immune against all commercially available antibiotics. It is evident that in order to prevent potential epidemic outbreaks of infectious diseases, a renewed focus on antibiotic research is highly desired. The group of lantibiotics has much promise as a new line of defense against pathogenic bacteria and many members are under evaluation for human applications. Currently, no methods exist for in depth medicinal chemistry on these compounds to improve their pharmacological properties. This research program seeks to establish technology that will allow doing this relying on the biosynthetic machinery. We have previously shown the power of this approach for the lantibiotics nisin, lacticin 481 and haloduracin and propose studies to better understand the enzymes involved in this grant application. Furthermore, we will extend our work to new promising lantibiotics and will investigate the use of the lantibiotic enzymes for applications other than lantibiotic engineering.